The chromatic photoreceptor neurons, R7 and R8, project their axons to the M3 and M6 layers of the medulla neuropil, respectively, and each pair of R7 and R8 axons innervates a single medulla column. Together these axonal termini tile the entire medulla neuropil. These organizing features, called layer-specific targeting and retinotopic mapping, are the hallmark of all complex visual systems. We have shown previously that during development, R7 and R8 axons project to specific layers and columns in two distinct stages. The sequential targeting of R7 and R8 axons to columns and layers reduces the number of potential synaptic partners (from over sixty medulla neurons to several), effectively reducing wiring complexity. By combining forward genetic screens and behavioral assays, we identified a number of connectivity mutants. We are currently focusing on one locus called overshoot, which represents a new class of layer-specific targeting phenotype: the synaptic terminals of mutant R7s extend small blob-like structures beyond their normal target layer into a deeper layer. Developmental analyses revealed that the overshoot phenotype appears during synaptogenesis around 80 hours after pupal formation. At this stage, wild-type R7 growth cones begin to retract filopodia from deeper layers and form synapses only in their correct target layer while mutant R7s fail to do so and form ectopic synapses in deeper layers. We carried out positional cloning and found that overshoot is an allele of Dynamitin (Dmn). Dmn is a component of the Dynactin complex which recruits cargos to Dynein motor in retrograde axonal transport. Western blot analysis revealed that overshoot is a hypomorphic allele, which produces approximately 70% of the wild-type protein level in the eyes. It has been suggested that a precise level of Dmn protein is critical for the integrity of the Dynactin complex. Consistent with this notion, over-expression or RNAi knock-down of Dmn in photoreceptors phenocopies overshoot. As no null Dmn allele is available, we generated, by P-element-mediated imprecise excision, a Dmn null mutation, which failed to complement overshoot. Unlike the viable overshoot mutants, the Dmn null mutation is embryonic lethal when rendered homozygous. Dmn null mutant R7s exhibited enlarged synaptic boutons that accumulate presynaptic protein Brp24. EM and light microscopic studies revealed that Dmn null mutant R7 terminals contain membrane-bound organelles, such as ER and Golgi, which are largely absence in adult wild-type terminals. We propose that Dmn is required for transporting ER/Golgi from growth cones to cell bodies during development and that perturbing this process indirectly causes the failure to retract neural processes during synaptogenesis.